Transcriptional regulator

ABSTRACT

A database search using the sequence of transcriptional regulator “RING3” having a bromodomain identified an EST sequence that is highly homologous with RING3. Using primers prepared based on the EST sequence, a gene encoding a novel transcriptional regulator having a bromodomain has been successfully isolated from a human testis cDNA library by polymerase chain reactions. The results of analysis of the isolated gene revealed that this gene is expressed strongly in testis cells with a potent proliferative ability. The use of the above transcriptional regulator and its gene enables screening candidate compounds for factors interacting with the transcriptional regulator or drugs controlling the activity of the regulator.

This application is a continuation-in-part of PCT/JP98/01782, filed Apr.17, 1998, and claims priority from Japanese Application No. 9/116402,filed Apr. 18, 1997.

TECHNICAL FIELD

The present invention relates to a novel transcriptional regulatorcontaining a bromodomain and a gene encoding it.

BACKGROUND ART

The bromodomain is a characteristic motif of amino acids found intranscriptional regulators, and is believed to be involved ininteraction with other proteins, such as other transcriptionalregulators. Proteins having a bromodomain usually contain one or two(Tamkun, J. W. et al., (1992) Cell, 68:561-572, Haynes, S. R. et al.,(1992) Nuc. Acids Res., 20:2603), but sometimes as many as fivebromodomain motifs (Nicolas, R. H. and Goodwin, G. H. (1996), Gene,175(12):233-240). This motif is found in a wide variety of animals. Forexample, it is identified in yeast (Winston, F. et al., (1987),Genetics, 115:649-656; Laurent, B. C. et al., (1991), Proc. Natl. Acad.Sci. USA, 25 88:2687-2691), Drosophila (Digan, M. E. et al., (1986),Dev. Biol., 114:161-169; Tamkun, J. W. et al., (1992), Cell,68:561-572), and mammals (Denis, G. V. and Green, M. R. (1996), Genesand Devel., 10:261-271; Yang, X. J. et al., (1996), Nature,382:319-324).

All transcriptional regulators containing a bromodomain serve to controlsignal-dependent transcription in actively proliferating cells (Tamkun,J. W. et al., (1992), Cell, 68:561-572; Haynes, S. R. et al., (1992),Nuc. Acids Res., 20:2603). Due to this feature, it is suggested thatcancer may develop if the gene for the protein containing a bromodomainis not normally controlled. In fact, several studies have shown thathuman transcriptional regulators with a bromodomain RING3, p300/CBP, andPCAF may be involved in oncogenesis.

RING3 was identified during an extensive analysis of the sequences ofhuman class II major histocompatibility systems (Beck et al., (1992) DNASeq. 2:203-210). The protein encoded by RING3 is homologous to D26362, ahuman gene (Nomura et al., (1994) DNA Res. 1:223-229) and fsh, adrosophila gene (Digan et al., (1986), Dev. Biol., 114: 161-169). Allthree genes encode proteins contain two copies of a bromodomain and aPEST sequences. The bromodomain is a motif consisting of 59 to 63 aminoacid residues and is considered to be involved in protein-proteininteractions. It is found among the transcriptional regulator proteins(Tamkun, J. W. et al., (1992) Cell, 68:561-572; Haynes, S. R. et al.,(1992) Nuc. Acids Res. 20:2603). The PEST sequence is a cluster ofproline (P), glutamic acid (E), serine (S) and threonine (T), whichcharacterizes the proteins that undergo rapid proteolysis in the cell.

The protein encoded by RING3 has a molecular weight of 90 kD and hasserine-threonine activity (Denis and Green, (1996) Genes and Devel.10:261-271). Comparison of the sequences of RING3 and fsh with those ofkinase domains of known serine-threonine kinases revealed that thesub-domains of the kinase motif are conserved, though in no particularorder (most of them are similar to the corresponding sub-domains of aproto-oncogene c-mos). Kinase activity of RING3 is stimulated byinterleukin-1 (IL-1) and forskolin, but not by a certain category ofcytokines (Denis and Green, (1996) Genes and Devel. 10:261-271). A closerelationship between kinase activity and growth phase in chronic andacute lymphocytic leukemia suggests the role RING3 plays in theleukemogenesis regulatory pathway (Denis and Green, (1996) Genes andDevel. 10:261-271). The analysis of the drosophila fsh gene suggestedthe interaction with the trithorax transcriptional regulator, a possibletarget for the putative phosphorylative activity of fsh (Digan et al.,(1986) Dev. Biol. 114:161-169; Mozer and Dawid, (1989) Proc. Natl. Acad.Sci. USA 86:3738-3742). The triathorax gene and its homologue ALL-1 havea C4HC3 zinc finger, a motif commonly found among the genes present atleukemia breakpoints(Aasland et al., (1995) Trends Biochem. Sci.20:56-59; Saha et al., (1995) Proc. Natl. Acad. Sci. USA 92:9737-9741).

In addition to RING3, at least two other bromodomain proteins, p300/CBPand PCAF, are associated with oncogenesis. Although p300 protein and CBPprotein are encoded by different genes, they are extremely closelyrelated, and therefore, they are often called p300/CBP. Mutations in CBPare often found in familial and sporadic cancers. Mutations in CBPsometimes result in Rubinstein-Taybi syndrome, which causes patients todevelop various malignant tumors (Petrij et al., (1995) Nature376:348-51). Furthermore, CBP is fused with MOZ at t(8;6)(p11;p13)translocation (Borrow et al., (1996) Nature Genet. 14:33-41). Thisfusion protein possibly causes leukemogenesis by its aberrantacetyltransferase activity (Brownwell and Allis, (1996) Curr. Opin.Genet. Devel. 6:176-184). Mutation in p300 is found in sporadic colonand gastric cancers (Muraoka et al., (1996) Oncogene 12:1565-1569), andp300 has been suggested to be a gene for a tumor-suppressing factorlocated on chromosome 22q. Another fact that suggests the role ofp300/CBP in cancer is that it interacts with the known oncogenes. Forexample, it is a co-activator of c-Myb (Dai, et al., (1996) Genes andDevel. 10:528-540) and c-Fos (Bannister and Kouzarides, (1996) Nature384:641-643) transcriptional factors, to which the E1A protein ofAdenovirus bind (Yang et al., (1996) Nature 382:319-324). Theinteraction between E1A and p300/CBP is inhibited by PCAF, a bromodomainprotein.

Like p300/CBP, PCAF also has histone acetyltransferase activity. PCAF,when exogenously expressed, can reduce the proliferation associated withE1A in cultured cells (Yang et al., (1996) Nature 382:319-324).Therefore one of the first mechanisms of the activity of the E1Aoncogene may be to inhibit the interaction between PCAF and p300.

Thus, it is thought that aberrant regulation of transcriptionalregulators containing a bromodomain may be closely related to variousdiseases, for example, cancer. Transcriptional regulators containing abromodomain have thus recently received much attention as novel targetsfor treating cancer.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to provide a noveltranscriptional regulator having a bromodomain and DNA encoding saidtranscriptional regulator. Another objective of the present invention isto provide a vector carrying said DNA, a transformant retaining saidDNA, and a process for producing a recombinant protein by utilizing saidtransformant. A further objective of the present invention is to providea method of screening a compound that binds to said transcriptionalregulator.

To solve the problems described above, the inventors searched a databasebased on the sequence of RING3 transcriptional regulator with abromodomain and found several EST sequences highly homologous to RING3.Primers were then prepared based on one of the EST sequences, andpolymerase chain reaction was performed in a human testis cDNA libraryusing the primers. As a result, a gene encoding a novel transcriptionalregulator with a bromodomain was successfully isolated. By analyzing theexpression of this gene, the inventors discovered that the gene ishighly expressed in the testis cells. Moreover, the inventors found thata factor interacting with the transcriptional regulator, or a candidatepharmaceutical compound that regulates activity of the transcriptionalregulator, can be screened by utilizing the transcriptional regulatorand the gene encoding it.

Thus, the present invention relates to novel transcriptional regulatorseach having a bromodomain and the genes encoding them, and to a methodof screening for a related factor or a candidate compound as amedicament using said proteins or genes, and more specifically relatesto:

(1) a transcriptional regulator having a bromodomain, which comprisesthe amino acid sequence shown in SEQ ID NO:1, or said sequence whereinone or more amino acids are substituted, deleted, or added;

(2) a transcriptional regulator having a bromodomain, which is encodedby DNA hybridizing with DNA comprising the nucleotide sequence shown inSEQ ID NO:2;

(3) DNA coding for the transcriptional regulator according to (1) or(2);

(4) a vector comprising the DNA according to (3);

(5) a transformant expressibly retaining the DNA according to (3);

(6) a method for producing the transcriptional regulator according to(1) or (2), which comprises culturing the transformant according to (5);

(7) an antibody binding to the transcriptional regulator according to(1) or (2);

(8) a method of screening a compound having binding activity to thetranscriptional regulator according to (1) or (2), wherein the methodcomprises

(a) contacting a sample with the transcriptional regulator according to(1) or (2) and

(b) selecting a compound having binding activity to the transcriptionalregulator according to (1) or (2);

(9) a compound having binding activity to the transcriptional regulatoraccording to (1) or (2), which can be isolated according to the methodof (8);

(10) the compound according to (9), which is naturally occurring; and

(11) DNA specifically hybridizing with DNA comprising the nucleotidesequence shown in SEQ ID NO:2 and having at least 15 nucleotides.

Here, the term “transcriptional regulator(s)” means protein(s) thatcontrol gene expression, and “bromodomain” means an amino acid motifconserved among the transcriptional regulators associated withsignal-dependent transcription, wherein said motif is involved inprotein-protein interaction.

The present invention relates to a novel transcriptional regulatorhaving a bromodomain. The amino acid sequence of the protein designatedTSB contained in the transcriptional regulator of the present inventionis shown in SEQ ID NO:1, and the nucleotide sequence of the cDNAencoding the protein is shown in SEQ ID NO:2. TSB protein is generallyknown as a region involved in interaction with other factors includingtranscriptional regulators, and it has a bromodomain(s) (amino acidpositions 49-109 and 292-352 of SEQ ID NO:1), a characteristic motif oftranscriptional regulators involved in cancer (FIG. 1). It is alsohighly expressed in the testis cells (Example 4). These facts suggestthat TSB protein, like other transcriptional regulators havingbromodomains, may be involved in cell proliferative diseases andcancers, particularly in testis cancer. In this connection, bromodomainsare thought to play an important role. Thus, TSB protein, or atranscriptional regulator functionally equivalent thereto, can be usedto prevent and treat cell proliferative diseases and cancers.

The transcriptional regulators of the present invention can be preparedas recombinant proteins generated using a recombinant gene technique, oras naturally-occurring proteins. The transcriptional regulators of thepresent invention include both recombinant and naturally-occurringproteins. The recombinant proteins can be prepared using a method suchas incorporating DNA encoding a transcriptional regulator of the presentinvention as described below (e.g., DNA having the nucleotide sequenceshown in SEQ ID NO:2) into a suitable expression vector, which is thenintroduced into host cells, and purifying the protein obtained from thetransformant. The naturally occurring proteins can be prepared using amethod such as preparing a column which utilizes an antibody obtainedfrom a small animal immunized with the recombinant protein prepared asabove or its partial peptide, and subjecting the extract from a tissueor cells in which a transcriptional regulator of the present inventionis overexpressed (e.g., testis) to affinity chromatography using saidcolumn.

The present invention also relates to transcriptional regulatorsfunctionally equivalent to the transcriptional regulators of the presentinvention. A method of introducing mutation into amino acids of aprotein to isolate such a protein is well known to one skilled in theart. Thus, it is well within the art of an ordinarily skilled person toisolate a protein functionally equivalent to the TSB protein having theamino acid sequence shown in SEQ ID NO:1 by appropriately modifying, forexample, substituting amino acids without affecting the function of theprotein using a site-directed mutagenesis system using PCR (GIBCO-BRL,Gaithersburg, Md.), a site-directed mutagenesis using oligonucleotides(Kramer, W. and Fritz, H. J. (1987), Methods in Enzymol., 154:350-367),or the similar methods. Mutation in an amino acid of a protein can alsooccur spontaneously. The transcriptional regulators of the presentinvention include those having the amino acid sequence (SEQ ID NO:1) ofthe TSB potein in which one or more amino acids are substituted,deleted, or added, and functionally equivalent to the TSB protein. Thenumber of mutagenized amino acids is not particularly limited as long asit retains function equivalent to the TSB proiten. It is usually 50amino acids or less, preferably 30 amino acids or less, more preferably10 amino acids or less, and most preferably five amino acids or less.

As another method of isolating a functionally equivalent proteinutilizing a hybridization technique (Sambrook, J. et al., MolecularCloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is wellknown to one skilled in the art. Based on the DNA sequence encoding theTBS protein (SEQ ID NO:2), or the fragment thereof, a person withordinary skill in the art can isolate DNA highly homologous to said DNAsequences using a hybridization technique (Sambrook, J. et al.,Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press,1989) to obtain a protein functionally equivalent to the TBS protein.The transcriptional regulators of the present invention include thoseencoded by DNA that hybridizes with DNA encoding the TBS protein andfunctionally equivalent to the TBS protein. Source organisms used toisolate a functionally equivalent protein includes, for example, mouse,rat, cattle, monkey, and pig as well as human. The hybridization andwashing conditions for isolating DNA encoding a functionally equivalentprotein are defined as low stringency: 42° C., 2×SSC, 0.1% SDS; moderatestringency: 50° C., 2×SSC, 0.1% SDS; and high stringency: 65° C., 2×SSC,0.1% SDS. The higher the temperature, the more highly homologous DNAwill be obtained. High amino acid homology means usually 40% or more,preferably 60% or more, more preferably 80% or more, or most preferably95% or more. The transcriptional regulator obtained by the hybridizationtechnique preferably contains bromodomain(s). It can also contain aserine/threonine kinase domain which functions to phosphorylate otherproteins, PEST sequence which is a characteristic sequence of theproteins undergoing rapid intracellular proteolysis, and a nucleartransport signal which functions to tranport the protein into nucleus.The presence of the bromodomain in the protein can be identified bysearching the bromodomain motif PROSITE database on DNASIS (HITACHISoftware engineering).

The present invention also relates to DNA encoding a protein of thepresent invention. The DNA of the present invention includes cDNA,genomic DNA, and chemically synthesized DNA, but is not limited theretoas long as it codes for a protein of the present invention. cDNA can beprepared, for example, by designing a primer based on the nucleic acidsequence shown in SEQ ID NO:2 and performing plaque PCR (see Affara, N.A. et al. (1994), Genomics, 22:205-210). The genomic DNA can be preparedaccording to a standard technique using, for example, Qiagen genomic DNAkits (Qiagen, Hilden, Germany). The DNA sequence thus obtained can bedetermined according to a standard technique using a commerciallyavailable dye terminator sequencing kit (Applied Biosystems) and thelike. In addition to applying to the production of recombinant proteinsas described below, the DNA of the present invention may be applied togene therapy and the like.

The present invention also relates to a vector into which the DNA of thepresent invention is inserted. The vector of the present inventionincludes various types of vectors, e.g. for expressing the protein ofthe present invention in vivo and for preparing recombinant proteins andappropriately selected depending on the purpose. Vectors used forexpressing the protein of the present invention in vivo (in particular,for gene therapy) include the adenovirus vector pAdexLcw and theretrovirus vector pZIPneo. An expression vector is particularly usefulfor producing a protein of the present invention. Although there is noparticular limitation to the expression vectors, the following vectorsare preferred: pREP4 (Qiagen, Hilden, Germany) when E. coli is used;SP-Q01 (Stratagene, La Jolla, Calif.) when yeast is used; and BAC-to-BACbaculovirus expression system (GIBCO-BRL, Gaithersburg, Md.) when insectcells are used. A LacSwitch II expression system (Stratagene; La Jolla,Calif.) is advantageous when mammalian cells, such as CHO, COS, andNIH3T3 cells, are used. The DNA of the present invention can be insertedinto vectors using a standard method.

The present invention also relates to a transformant expressiblyretaining the DNA of the present invention. The transformants of thepresent invention include one harboring the above-described vector intowhich the DNA of the present invention is inserted and one having theDNA of the present invention integrated into its genome. The DNA of thepresent invention can be retained in the transformant in any form aslong as the transformant expressibly retains the DNA of the presentinvention. There is no limitation to host. cells into which a vector ofthe present invention is introduced. If the cells are used to express aprotein of the present invention for the purpose of ex vivo genetherapy, various cells can be used as target cells suited to diseases.Cells such as E. coli, yeast cells, animal cells, and insect cells canbe used for producing the protein of the present invention. The vectorcan be introduced into the cells by methods such as electroporation andthe calcium phosphate method. Recombinant proteins can be isolated andpurified from the transformants generated for producing the saidproteins according to a standard method.

The present invention also relates to antibodies that bind to thetranscriptional regulators of the present invention. The antibodies ofthe present invention include, but are not limited to, polyclonal andmonoclonal antibodies. Also included are antisera obtained by immunizingan animal such as a rabbit with a protein of the present invention, anyclass of polyclonal or monoclonal antibodies, humanized antibodiesgenerated by gene recombination, and human antibodies. The antibodies ofthe present invention can be prepared according to the following method.For polyclonal antibodies, antisera can be obtained by immunizing asmall animal, such as a rabbit, with a transcriptional regulator of thepresent invention or a partial peptide thereof, then recovering thefractions that only recognize the transcriptional regulator of thepresent invention through an affinity column coupled with thetranscriptional regulator of the present invention. Immunoglobulin G orM can be prepared by purifying the fractions through a Protein A or Gcolumn. For monoclonal antibodies, a small animal, such as a mouse, isimmunized with a transcriptional regulator of the invention, the spleenis removed from the mouse and homogenized into cells, the cells arefused with myeloma cells from a mouse using a reagent such aspolyethylene glycol, and clones that produce antibodies against thetranscriptional regulator of the invention are selected from theresulting fused cells (hybridoma). The hybridoma obtained is thentransplanted into the abdominal cavity of a mouse, and the ascites arerecovered from the mouse. The obtained monoclonal antibodies can then beprepared by purifying, for example, by ammonium sulfate precipitationthrough a Protein A or G column, by DEAE ion exchanging chromatography,or through an affinity column coupled with the protein of the invention.Besides being used to purify or detect the transcriptional regulators ofthe present invention, the antiobodies of the present invention canbeused as a drug for suppressing the functions of the transcriptionalregulator of the present invention. When an antibody is used as a drug,a human or humanized antibody is effective with regard toimmunogenicity. A human or humanized antibody can be prepared accordingto methods well known in the art. For example, a human antibody can beprepared by immunizing a mouse whose immune system is replaced by ahuman system with the transcriptional regulator of the presentinvention. A humanized antibody can be prepared by the CDR graftingmethod in which an antibody gene is cloned from monoclonalantibody-producing cells and its antigenic determinant site istransplanted to an existing human antibody.

The present invention also relates to a method for screening a compoundthat binds to transcriptional regulators of the present invention. Thescreening method of the present invention includes steps of (a)contacting a transcriptional regulator of the present invention with atest sample and (b) selecting a compound that has binding activity forthe transcriptional regulator of the present invention. Test samplesused for the screening include, but are not limited to, a library ofsynthetic low molecular weight compounds, a purified protein, anexpression product of a gene library, a library of synthetic peptides acell extract, and a supernatant of the cell culture. Various methodswell known to one skilled in the art can be used for selecting acompound binding to a transcriptional regulator of the presentinvention.

A protein that-binds to a transcriptional regulator of the presentinvention can be screened by West-western blotting comprising steps ofgenerating a cDNA library from the tissues of cells expected to expressthe protein that binds to a transcriptional regulator of the presentinvention (e.g., testis) using a phage vector (λgt11, ZAPII, etc.),allowing the cDNA library to express on the LB-agarose plate, fixing theexpressed proteins on a filter, reacting them with the transcriptionalregulator of the present invention purified as a biotin-labeled proteinor a fusion protein with GST protein, and detecting plaques expressingthe protein bound to the regulator on the filter with streptavidin oranti-GST antibody (Skolnik, E. Y., Margolis, B., Mohammadi, M.,Lowenstein, E., Fisher, R., Drepps, A., Ullrich, A. and Schlessinger, J.(1991), Cloning of PI3 kinase-associated p85 utilizing a novel methodfor expression/cloning of target proteins for receptor tyrosine kinases,Cell, 65:83-90). Alternatively, the method comprises expressing in yeastcells a transcriptional regulator of the present invention which isfused with SFR or GAL4 binding region, constructing a cDNA library inwhich proteins are expressed in a fusion protein with the transcriptionactivation site of VP16 or GAL4 from the cells expected to express aprotein that binds to the transcriptional regulator of the presentinvention, introducing the cDNA library into the above-described yeastcells, isolating the cDNA derived from the library from the detectedpositive clones, and introducing and expressing it in E. coli. (If aprotein that binds to the transcriptional regulator of the presentinvention is expressed, a reporter gene is activated by the binding ofthe two proteins. The positive clones can then be identified.) Thismethod can be performed using Two-hybrid system (MATCHMAKER Two-HybridSystem, Mammalian MATCHMAKER Two-Hybrid Assay Kit, MATCHMAKER One-HybridSystem (all from Clontech); HybriZAP Two-Hybrid Vector System(Stratagene) or in accordance with Dalton, S. and Treisman R. (1992),Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element, Cell, 68:597-612). Another methodis to apply a culture supernatant or a cell extract from the cellssuspected to express a protein which binds to the transcriptionalregulator of the present invention onto an affinity column coupled withthe transcriptional regulator of the present invention, and purify theprotein specifically bound to the column.

Also well known to one skilled in the art are a method of screeningmolecules that bind to a transcriptional regulator of the presentinvention by reacting the immobilized transcriptional regulator ofpresent invention with a synthetic compound, natural substance bank, ora random phage peptide display library, and a method of isolating lowmolecular weight compounds, proteins (or their genes), or peptides whichbind to a transcriptional regulator of the present invention byutilizing the high-throughput technique of combinatorial chemistry(Wrighton, N. C., Farrell, F. X., Chang, R., Kashuyap, A. K., Barbone,F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K.,Dower, W. J., Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) Jul. 26, 1996, 273:458-464;Verdine, G. L., The combinatorial chemistry of nature, Nature (ENGLAND),Nov. 7, 1996, 384:11-13; Hogan, J. C. Jr., Directed combinatorialchemistry, Nature (ENGLAND), Nov. 7, 1996, 384:17-19). The compoundsthus isolated by the screening method of the present invention arecandidates for drugs for enhancing or surpressing the activity of atranscriptional regulator of the present invention. When the compoundsisolated by the screening method of the present invention are used aspharmaceuticals, they can be formulated by a known pharmacologicalprocess. For example, they can be administered to a patient withpharmaceutically acceptable carriers and vehicles (e.g., physiologicalsaline, vegetable oil, a dispersant, a surfactant, and a stabilizer).The compounds may be percutaneously, intranasally, transbronchially,intramuscularly, intravenously, or orally administered, depending ontheir properties.

The present invention also relates to DNA specifically hybridizing withDNA coding the TBS protein and having at least 15 nucleotides. As usedherein, “specifically hybridizing” means that no cross-hybridizationoccurs between DNA encoding other proteins under conditions of moderatestringency. Such DNA may be used as a probe for detecting and isolatingthe DNA encoding the TBS protein, and as a primer for amplifying the DNAencoding the protein of the present invention. Specific examples of theprimers include those shown in SEQ ID NOs:3 and 4.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, and(iii) cell clones: e.g., as these occur in a DNA library such as a cDNAor genomic DNA library.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological compounds, such as those in cellular material,,viralmaterial, or culture medium, with which the polypeptide was associated(e.g., in the course of production by recombinant DNA techniques orbefore purification from a natural biological source). The substantiallypure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%)pure by dry weight. Purity can be measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A “conservative amino acid substitution” is one in which an amino acidresidue is replaced with another residue having a chemically similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

As used herein, “percent identity” of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul(Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlinand Altschul (Proc. Natl. Acad. Sci. USA 90:4873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequence homologous to a nucleicacid molecules of the invention. BLAST protein searches are performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to a reference polypeptide. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25;3389-3402, 19971).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used. Theseprograms can be found the World Wide Web at the web site of the NationalCenter for Biotechnology Information NCBI).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions, will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The materials, methods,and examples are illustrative only and not intended to be limiting.Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequence (SEQ ID NO:2) of TSB aligned withthe amino acid sequence (SEQ ID NO:1) of the open reading frame. Thethree motifs identified by the search of the PROSITE database areunderlined. Two bromodomains (amino acid positions 49-109 and 292-352)and a PEST sequence (amino acid positions 636-672) are identified.

FIG. 2 compares the amino acid sequences of the predicted kinase domainsof TSB (SEQ ID NOs:5-12), RING3 (SEQ ID NOs: 13-20), and fsh (SEQ IDNOs:21-28). The sub-domains of kinase are disclosed in Denis and Green,(1996) Genes and Devel. 10:261-271, and sub-domains I-II are excluded.The numerals correspond to the translation position of TSB. Theconserved residues are shaded. adjacent markers on chromosome 1pdetermined by radiation hybrid analysis.

FIG. 3 shows the map location of TSB. The position is indicated relativeto the positions of the adjacent markers on the chromosome 1p determinedby radiation hybrid analysis.

FIG. 4A shows the results of Northern blot analysis of TSB in the normaltissues (Lane 1, heart; Lane 2, brain; Lane 3, placenta; Lane 4, lung;Lane 5, liver; Lane 6, skeletal muscle; Lane 7, kidney; Lane 8,pancreas; Lane 9, spleen; Lane 10, thymus; Lane 11, prostate; Lane 12,testis; Lane 13, ovary; Lane 14, small intestine; Lane 15, colon (mucouslining); and Lane 16, peripheral leukocytes).

FIG. 4B shows the results of Northern blot analysis of TSB in carcinomacell lines (Lane 1, promyelocytic leukemia HL-60; Lane 2, HeLa S3 cells;Lane 3, chronic myelocytic leukemia K-56; Lane 4, lymphoblastic leukemiaMOLT-4; Lane 5, Burkitt's lymphoma Raji; Lane 6, large intestinaladenocarcinoma SW480; Lane 7, lung carcinoma S549; and Lane 8, melanomaG361). The positions of molecular weight markers are indicated on theright.

BEST MODE FOR IMPLEMENTING THE INVENTION

The following examples illustrate the present invention in more detail,but are not to be construed to limit the scope of the present invention.

EXAMPLE 1 Identification of EST Homologous to RING3 and Isolation of itsFull-length Sequence

A BLAST search of the EST database identified numerous ESTs homologousto the DNA sequence of the RING3 gene (Beck et al., (1992), DNA Seq.2:203-210) used as a probe. Among these ESTs, EST H64204, which wasderived from a testis- specific cDNA library (Diatchenko et al., (1996)Proc. Natl. Acad. Sci. USA 93:6025-6030), had 65% sequence homology toRING3 spanning 285 bp.

To clone the full-length sequence of EST H64204, PCR primers U(AATGTCTCTGCCAAGTCGACAA; SEQ ID NO:3) and L (AGCATCCACAGGACGTTGAAAG; SEQID NO:4) were designed to perform PCR using testis cDNAs as templates.PCR was carried out at 94° C. for 8 min. followed by 35 cycles of 94° C.for 30 sec (heat annealing), 60° C. for 1 min (annealing), and 72° C.for 1 min (extension). AmpliTaq gold (Perkin Elmer) was used as anenzyme for PCR. This resulted in a PCR product of 175 bp. Subsequently,a testis cDNA library was screened (Clontech; HL3024a) using this PCRproduct as a probe. All the probes were labeled with [−³²P]dCTP byrandom priming and were purified through Chromaspin (10 columns;Clontech). Hybridization was performed in ExpressHyb hybridizationsolution (Clontech) for 1 hour at 65°. The filter was washed at thefinal stringency of 1×SSC, 0.1% SDS, 65° C. The sequence of a cDNA clonethat was aligned with the EST sequence was used to re-screen thelibrary. This process was repeated until a series of overlapped clonesgiving a full-length sequence of the coding region of the gene wasobtained. As a result, a continuous sequence of 3,104 bp encoding 947amino acids was found in an open reading frame (ORF) at the nucleotidepositions 106-2946. The ORF is followed by a short stretch of a 3′untranslated region of 60 bp, which is terminated with a poly(A) tailthat has a polyadenylated signal (ATTAAA) in the 20 bp upstream of it.The inventors designated the isolated clone “TSB (testis specificbromodomain). SEQ ID NO:2 shows the nucleotide sequence of TSB alongwith the predicted amino acid sequence thereof. The predicted amino acidsequence is also shown in SEQ ID NO:1. The nucleotide sequences weredetermined with the automated sequencer ABI377 (Perkin Elmer) by use ofABI dye terminator chemistry.

EXAMPLE 2 Homology and Motifs

RING3 (66% homology, 59% identity spanning 649 amino acids), D26362 (62%homology, 56% identity spanning 649 amino acids), and fsh (62% homology,56% identity spanning 565 amino acids) were identified as the amino acidsequences having the highest homology with TBS by searching proteindatabases.

Two bromodomains (amino acid positions 49-109 and 292-352) wereidentified by searching the PROSITE database for the motif of amino acidsequence. A PEST sequence (Rodgers et al., (1986) Science 234:364) wasalso present in amino acid positions 636-672. FIG. 1 shows the locationsof these motifs.

Since RING3 is also known to have serine-threonine kinase activity(Denis and Green, (1996) Genes and Devel. 10:261-271), the amino acidsequence of TSB was compared with that of the predicted kinase domain ofRING3 using Bestfit at GCG. The result showed that the predicted kinasedomain of RING3 is very well conserved in TSB (FIG. 2). However, it wasfound that TBS did not contain sub-domain I, which codes for thepredicted ATP-binding domain, and sub-domain II, which codes forcatalytic lysine, suggesting that the kinase activity of TSB waspossibly lost.

In addition, since the RING3 protein is known to be localized in thenucleus, the predicted nuclear transport signal of TSB was identifiedusing the PSORT program. As a result, four copies of the nucleartransport signal (in the 488, 489, 575 and 919 positions), each copyconsisting of four residues, and two copies of Robins and Dingwallconsensus sequence (Robins and Dingwall, (1991) Cell 64:615-23) (in the445 and 603 positions) were discovered. Thus, like RING3, nuclearlocalization of TSB was also indicated.

EXAMPLE 3 Mapping of TSB

To identify the locus of TSB, DNA from 24 human/rodent singlechromosomal somatic cell lines obtained from Coriell Cell Repositories,New Jersey (Dubois and Naylor, (1993) Genomics 16:315-319) wereamplified using primers U (SEQ ID NO:3) and L (SEQ ID NO:4).

A panel of single chromosomal hybrid cell lines was screened for theTSB-localized region, using primers U (SEQ ID NO:3) and L (SEQ ID NO:4).As a result, the predicted product of 175 bp was amplified only in thecell line GM 13139, a single chromosomal cell line for humanchromosome 1. The same primers were used for PCR for a GeneBridge4radiation hybrid panel (Walter et al., (1994) Nature Genetics 7:22-28).The binary codes generated by assessing each hybrid as positive ornegative for the amplification were compared with the analogous codesfor the markers constituting the framework map, using the server locatedon the World Wide Web at the web site of the Whitehead Institute forBiomedical Research/MIT Center for Genome Research. This procedure wasrepeated to give independent scores. The two experiments yieldedidentical binary codes, and TSB was shown to be located on chromosome lpbetween markers WI-7719 and WI-3099 (D1S2154) (FIG. 3).

EXAMPLE 4 Analysis of TSB Expression

Northern analysis of 16 normal tissues was conducted using the probeprepared by PCR amplification of the testis cDNAs using primers U (SEQID NO:3) and L (SEQ ID NO:4). The probe strongly hybridized with mRNA of3.5 kb and weakly hybridized with that of 4.0 kb (FIG. 4A). This resultwas consistent with the source testis-specific library of the EST usedto identify TSB (Diatchenko et al., (1996) Proc. Natl. Acad. Sci. USA93:6025-6030). In addition, the probe cross-hybridized with the twospecies of mRNA (about 2.0 and 4.5 kb) commonly expressed in thesetissues. Since this probe contained the sequence encoding a bromodomain,the transcripts of the two species potentially represent otherbromodomain genes. Furthermore, a panel of mRNA derived from eight tumorcell lines was screened along with the panel of the normal tissues,since, several other testis-specific genes, in particular the MAGEfamily (van der Bruggen et al., (1991) Science 254:1643-1647) areexpressed in tumor tissues. However, expression of TSB was not detectedin any of the cell lines tested (FIG. 4B). Likewise, a panel of 10samples of lung cancer was negative for the TSB expression (data are notshown). The conditions for hybridization were the same as thosedescribed in Example 1.

INDUSTRIAL APPLICABILITY

The present invention provides a novel transcriptional regulator havinga bromodomain, DNA encoding said transcriptional regulator, a vectorcarrying said DNA, a transformant expressibly retaining said DNA, anantibody binding to said transcriptional regulator, and a method ofscreening a compound that binds to said transcriptional regulator. Thetranscriptional regulator of the present invention is considered to forma family together with a transcriptional regulator RING3 that is thoughtto be associated with cancer. It is abundantly expressed in the testis.Accordingly, the transcriptional regulator of the present invention andDNA encoding said transcriptional regulator can be used to screen fortherapeutics to treat diseases such as cell-proliferative diseases andcancer, particularly testis cancer; diseases attributed to aplasia anddysfunction of sperm; or for contraceptives. Antibodies and othercompounds that bind to the transcriptional regulator of the presentinvention can also be used as therapeutics.

28 1 947 PRT Homo sapiens 1 Met Ser Leu Pro Ser Arg Gln Thr Ala Ile IleVal Asn Pro Pro Pro 1 5 10 15 Pro Glu Tyr Ile Asn Thr Lys Lys Asn GlyArg Leu Thr Asn Gln Leu 20 25 30 Gln Tyr Leu Gln Lys Val Val Leu Lys AspLeu Trp Lys His Ser Phe 35 40 45 Ser Trp Pro Phe Gln Arg Pro Val Asp AlaVal Lys Leu Lys Leu Pro 50 55 60 Asp Tyr Tyr Thr Ile Ile Lys Asn Pro MetAsp Leu Asn Thr Ile Lys 65 70 75 80 Lys Arg Leu Glu Asn Lys Tyr Tyr AlaLys Ala Ser Glu Cys Ile Glu 85 90 95 Asp Phe Asn Thr Met Phe Ser Asn CysTyr Leu Tyr Asn Lys Pro Gly 100 105 110 Asp Asp Ile Val Leu Met Ala GlnAla Leu Glu Lys Leu Phe Met Gln 115 120 125 Lys Leu Ser Gln Met Pro GlnGlu Glu Gln Val Val Gly Val Lys Glu 130 135 140 Arg Ile Lys Lys Gly ThrGln Gln Asn Ile Ala Val Ser Ser Ala Lys 145 150 155 160 Glu Lys Ser SerPro Ser Ala Thr Glu Lys Val Phe Lys Gln Gln Glu 165 170 175 Ile Pro SerVal Phe Pro Lys Thr Ser Ile Ser Pro Leu Asn Val Val 180 185 190 Gln GlyAla Ser Val Asn Ser Ser Ser Gln Thr Ala Ala Gln Val Thr 195 200 205 LysGly Val Lys Arg Lys Ala Asp Thr Thr Thr Pro Ala Thr Ser Ala 210 215 220Val Lys Ala Ser Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Val 225 230235 240 Ala Leu Pro Pro Ile Lys Glu Asn Met Pro Lys Asn Val Leu Pro Asp245 250 255 Ser Gln Gln Gln Tyr Asn Val Val Glu Thr Val Lys Val Thr GluGln 260 265 270 Leu Arg His Cys Ser Glu Ile Leu Lys Glu Met Leu Ala LysLys His 275 280 285 Phe Ser Tyr Ala Trp Pro Phe Tyr Asn Pro Val Asp ValAsn Ala Leu 290 295 300 Gly Leu His Asn Tyr Tyr Asp Val Val Lys Asn ProMet Asp Leu Gly 305 310 315 320 Thr Ile Lys Glu Lys Met Asp Asn Gln GluTyr Lys Asp Ala Tyr Ser 325 330 335 Phe Ala Ala Asp Val Arg Leu Met PheMet Asn Cys Tyr Lys Tyr Asn 340 345 350 Pro Pro Asp His Glu Val Val ThrMet Ala Arg Met Leu Gln Asp Val 355 360 365 Phe Glu Thr His Phe Ser LysIle Pro Ile Glu Pro Val Glu Ser Met 370 375 380 Pro Leu Cys Tyr Ile LysThr Asp Ile Thr Glu Thr Thr Gly Arg Glu 385 390 395 400 Asn Thr Asn GluAla Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu 405 410 415 Asp Glu ArgVal Lys Arg Leu Ala Lys Leu Gln Glu Gln Leu Lys Ala 420 425 430 Val HisGln Gln Leu Gln Val Leu Ser Gln Val Pro Phe Arg Lys Leu 435 440 445 AsnLys Lys Lys Glu Lys Ser Lys Lys Glu Lys Lys Lys Glu Lys Val 450 455 460Asn Asn Ser Asn Glu Asn Pro Arg Lys Met Cys Glu Gln Met Arg Leu 465 470475 480 Lys Glu Lys Ser Lys Arg Asn Gln Pro Lys Lys Arg Lys Gln Gln Phe485 490 495 Ile Gly Leu Lys Ser Glu Asp Glu Asp Asn Ala Lys Pro Met AsnTyr 500 505 510 Asp Glu Lys Arg Gln Leu Ser Leu Asn Ile Asn Lys Leu ProGly Asp 515 520 525 Lys Leu Gly Arg Val Val His Ile Ile Gln Ser Arg GluPro Ser Leu 530 535 540 Ser Asn Ser Asn Pro Asp Glu Ile Glu Ile Asp PheGlu Thr Leu Lys 545 550 555 560 Ala Ser Thr Leu Arg Glu Leu Glu Lys TyrVal Ser Ala Cys Leu Arg 565 570 575 Lys Arg Pro Leu Lys Pro Pro Ala LysLys Ile Met Met Ser Lys Glu 580 585 590 Glu Leu His Ser Gln Lys Lys GlnGlu Leu Glu Lys Arg Leu Leu Asp 595 600 605 Val Asn Asn Gln Leu Asn SerArg Lys Arg Gln Thr Lys Ser Asp Lys 610 615 620 Thr Gln Pro Ser Lys AlaVal Glu Asn Val Ser Arg Leu Ser Glu Ser 625 630 635 640 Ser Ser Ser SerSer Ser Ser Ser Glu Ser Glu Ser Ser Ser Ser Asp 645 650 655 Leu Ser SerSer Asp Ser Ser Asp Ser Glu Ser Glu Met Phe Pro Lys 660 665 670 Phe ThrGlu Val Lys Pro Asn Asp Ser Pro Ser Lys Glu His Val Lys 675 680 685 LysMet Lys Asn Glu Cys Ile Leu Pro Glu Gly Arg Thr Gly Val Thr 690 695 700Gln Ile Gly Tyr Cys Val Gln Asp Thr Thr Ser Ala Asn Thr Thr Leu 705 710715 720 Val His Gln Thr Thr Pro Ser His Val Met Pro Pro Asn His His Gln725 730 735 Leu Ala Phe Asn Tyr Gln Glu Leu Glu His Leu Gln Thr Val LysAsn 740 745 750 Ile Ser Pro Leu Gln Ile Leu Pro Pro Ser Gly Asp Ser GluGln Leu 755 760 765 Ser Asn Gly Ile Thr Val Met His Pro Ser Gly Asp SerAsp Thr Thr 770 775 780 Met Leu Glu Ser Glu Cys Gln Ala Pro Val Gln LysAsp Ile Lys Ile 785 790 795 800 Lys Asn Ala Asp Ser Trp Lys Ser Leu GlyLys Pro Val Lys Pro Ser 805 810 815 Gly Val Met Lys Ser Ser Asp Glu LeuPhe Asn Gln Phe Arg Lys Ala 820 825 830 Ala Ile Glu Lys Glu Val Lys AlaArg Thr Gln Glu Leu Ile Arg Lys 835 840 845 His Leu Glu Gln Asn Thr LysGlu Leu Lys Ala Ser Gln Glu Asn Gln 850 855 860 Arg Asp Leu Gly Asn GlyLeu Thr Val Glu Ser Phe Ser Asn Lys Ile 865 870 875 880 Gln Asn Lys CysSer Gly Glu Glu Gln Lys Glu His Pro Gln Ser Ser 885 890 895 Glu Ala GlnAsp Lys Ser Lys Leu Trp Leu Leu Lys Asp Arg Asp Leu 900 905 910 Ala ArgPro Lys Glu Gln Glu Arg Arg Arg Arg Glu Ala Met Val Gly 915 920 925 ThrIle Asp Met Thr Leu Gln Ser Asp Ile Met Thr Met Phe Glu Asn 930 935 940Asn Phe Asp 945 2 3104 DNA Homo sapiens CDS (106)...(2946) 2 ggcaagatgttcctgggagg tcaagttaag agtcaaaaat aattcattag atttaacaat 60 ttagcatggacatgtacttg tagacaggat tcaaagcagt taaga atg tct ctg cca 117 Met Ser LeuPro 1 agt cga caa aca gct att att gtt aac cct cct cca cca gaa tat ata165 Ser Arg Gln Thr Ala Ile Ile Val Asn Pro Pro Pro Pro Glu Tyr Ile 5 1015 20 aat act aag aaa aat ggg cga ttg aca aat caa ctt cag tat cta caa213 Asn Thr Lys Lys Asn Gly Arg Leu Thr Asn Gln Leu Gln Tyr Leu Gln 2530 35 aaa gtt gtc cta aag gat tta tgg aag cat agt ttt tca tgg ccc ttt261 Lys Val Val Leu Lys Asp Leu Trp Lys His Ser Phe Ser Trp Pro Phe 4045 50 caa cgt cct gtg gat gct gtg aaa cta aag ttg cct gat tat tat acc309 Gln Arg Pro Val Asp Ala Val Lys Leu Lys Leu Pro Asp Tyr Tyr Thr 5560 65 att ata aaa aac cca atg gat tta aat aca att aag aag cgc ttg gag357 Ile Ile Lys Asn Pro Met Asp Leu Asn Thr Ile Lys Lys Arg Leu Glu 7075 80 aat aaa tat tat gcg aag gct tca gaa tgt ata gaa gac ttc aat aca405 Asn Lys Tyr Tyr Ala Lys Ala Ser Glu Cys Ile Glu Asp Phe Asn Thr 8590 95 100 atg ttc tca aat tgt tat tta tat aac aag cct gga gat gac attgtt 453 Met Phe Ser Asn Cys Tyr Leu Tyr Asn Lys Pro Gly Asp Asp Ile Val105 110 115 ctt atg gca caa gct cta gag aag ctg ttt atg cag aaa tta tctcag 501 Leu Met Ala Gln Ala Leu Glu Lys Leu Phe Met Gln Lys Leu Ser Gln120 125 130 atg cca caa gaa gag caa gtt gtg ggt gtt aag gaa aga atc aagaaa 549 Met Pro Gln Glu Glu Gln Val Val Gly Val Lys Glu Arg Ile Lys Lys135 140 145 ggc act caa cag aat ata gct gtt tct tct gct aaa gaa aaa tcatca 597 Gly Thr Gln Gln Asn Ile Ala Val Ser Ser Ala Lys Glu Lys Ser Ser150 155 160 ccc agc gca aca gaa aaa gta ttt aag cag caa gaa att cct tctgta 645 Pro Ser Ala Thr Glu Lys Val Phe Lys Gln Gln Glu Ile Pro Ser Val165 170 175 180 ttt cct aag aca tct att tct ccc ttg aac gtg gta cag ggagct tca 693 Phe Pro Lys Thr Ser Ile Ser Pro Leu Asn Val Val Gln Gly AlaSer 185 190 195 gtc aac tcc agt tca caa act gcg gcc caa gtt aca aaa ggtgtg aag 741 Val Asn Ser Ser Ser Gln Thr Ala Ala Gln Val Thr Lys Gly ValLys 200 205 210 agg aaa gca gat aca aca act cct gca act tca gca gtt aaagca agt 789 Arg Lys Ala Asp Thr Thr Thr Pro Ala Thr Ser Ala Val Lys AlaSer 215 220 225 agt gaa ttt tct cca aca ttc aca gaa aaa tca gtg gca ctgcca cct 837 Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Val Ala Leu ProPro 230 235 240 ata aaa gaa aat atg cca aag aat gtt ttg cca gat tct cagcaa caa 885 Ile Lys Glu Asn Met Pro Lys Asn Val Leu Pro Asp Ser Gln GlnGln 245 250 255 260 tat aat gtt gtg gag act gtt aaa gta act gaa caa ttaagg cac tgt 933 Tyr Asn Val Val Glu Thr Val Lys Val Thr Glu Gln Leu ArgHis Cys 265 270 275 agt gag att ctt aaa gaa atg ctt gca aag aaa cat ttttca tat gca 981 Ser Glu Ile Leu Lys Glu Met Leu Ala Lys Lys His Phe SerTyr Ala 280 285 290 tgg ccc ttt tat aat cct gtt gac gtt aat gct ttg ggactc cat aac 1029 Trp Pro Phe Tyr Asn Pro Val Asp Val Asn Ala Leu Gly LeuHis Asn 295 300 305 tac tat gac gtt gtc aaa aat ccg atg gat ctt gga actatt aag gag 1077 Tyr Tyr Asp Val Val Lys Asn Pro Met Asp Leu Gly Thr IleLys Glu 310 315 320 aaa atg gat aac caa gaa tat aag gat gca tac tca tttgcg gca gat 1125 Lys Met Asp Asn Gln Glu Tyr Lys Asp Ala Tyr Ser Phe AlaAla Asp 325 330 335 340 gtt aga tta atg ttc atg aat tgc tac aag tac aatcct cca gat cac 1173 Val Arg Leu Met Phe Met Asn Cys Tyr Lys Tyr Asn ProPro Asp His 345 350 355 gaa gtt gtg aca atg gca aga atg ctt cag gat gttttc gaa acg cat 1221 Glu Val Val Thr Met Ala Arg Met Leu Gln Asp Val PheGlu Thr His 360 365 370 ttt tca aag atc ccg att gaa cct gtt gag agt atgcct tta tgt tac 1269 Phe Ser Lys Ile Pro Ile Glu Pro Val Glu Ser Met ProLeu Cys Tyr 375 380 385 atc aaa aca gat atc aca gaa acc act ggt aga gagaac act aat gaa 1317 Ile Lys Thr Asp Ile Thr Glu Thr Thr Gly Arg Glu AsnThr Asn Glu 390 395 400 gcc tcc tct gaa ggg aac tct tct gat gat tct gaagat gag cga gtt 1365 Ala Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu AspGlu Arg Val 405 410 415 420 aag cgt ctt gca aag ctt cag gag cag ctt aaagct gta cat caa cag 1413 Lys Arg Leu Ala Lys Leu Gln Glu Gln Leu Lys AlaVal His Gln Gln 425 430 435 ctc cag gtt ttg tcc caa gta cct ttc cgt aagcta aat aaa aag aaa 1461 Leu Gln Val Leu Ser Gln Val Pro Phe Arg Lys LeuAsn Lys Lys Lys 440 445 450 gag aag tct aaa aag gaa aag aaa aaa gaa aaggtt aat aac agc aat 1509 Glu Lys Ser Lys Lys Glu Lys Lys Lys Glu Lys ValAsn Asn Ser Asn 455 460 465 gaa aat cca aga aaa atg tgt gag caa atg aggcta aag gaa aag tcc 1557 Glu Asn Pro Arg Lys Met Cys Glu Gln Met Arg LeuLys Glu Lys Ser 470 475 480 aag aga aat cag cca aag aaa agg aaa caa cagttc att ggt cta aaa 1605 Lys Arg Asn Gln Pro Lys Lys Arg Lys Gln Gln PheIle Gly Leu Lys 485 490 495 500 tct gaa gat gaa gat aat gct aaa cct atgaac tat gat gag aaa agg 1653 Ser Glu Asp Glu Asp Asn Ala Lys Pro Met AsnTyr Asp Glu Lys Arg 505 510 515 cag tta agt ctg aat ata aac aaa ctc cctgga gat aaa ctt ggg cga 1701 Gln Leu Ser Leu Asn Ile Asn Lys Leu Pro GlyAsp Lys Leu Gly Arg 520 525 530 gta gtt cac ata ata caa tca aga gag ccttct ctg agc aat tcc aat 1749 Val Val His Ile Ile Gln Ser Arg Glu Pro SerLeu Ser Asn Ser Asn 535 540 545 cct gat gag ata gag ata gac ttt gaa acactg aaa gca tca aca cta 1797 Pro Asp Glu Ile Glu Ile Asp Phe Glu Thr LeuLys Ala Ser Thr Leu 550 555 560 aga gaa tta gaa aaa tat gtt tcg gca tgtcta aga aag aga cca tta 1845 Arg Glu Leu Glu Lys Tyr Val Ser Ala Cys LeuArg Lys Arg Pro Leu 565 570 575 580 aaa cct cct gct aag aaa ata atg atgtcc aaa gaa gaa ctt cac tca 1893 Lys Pro Pro Ala Lys Lys Ile Met Met SerLys Glu Glu Leu His Ser 585 590 595 cag aaa aaa cag gaa ttg gaa aag cggtta ctg gat gtt aat aat cag 1941 Gln Lys Lys Gln Glu Leu Glu Lys Arg LeuLeu Asp Val Asn Asn Gln 600 605 610 tta aat tct aga aaa cgt caa aca aaatct gat aaa acg caa cca tcc 1989 Leu Asn Ser Arg Lys Arg Gln Thr Lys SerAsp Lys Thr Gln Pro Ser 615 620 625 aaa gct gtt gaa aat gtt tcc cga ctgagt gag agc agc agc agc agc 2037 Lys Ala Val Glu Asn Val Ser Arg Leu SerGlu Ser Ser Ser Ser Ser 630 635 640 agc agc tca tca gag tct gaa agt agcagc agt gac tta agc tct tca 2085 Ser Ser Ser Ser Glu Ser Glu Ser Ser SerSer Asp Leu Ser Ser Ser 645 650 655 660 gac agc agt gat tct gaa tca gaaatg ttc cct aag ttt aca gaa gta 2133 Asp Ser Ser Asp Ser Glu Ser Glu MetPhe Pro Lys Phe Thr Glu Val 665 670 675 aaa cca aat gat tct cct tct aaagag cat gta aag aaa atg aag aat 2181 Lys Pro Asn Asp Ser Pro Ser Lys GluHis Val Lys Lys Met Lys Asn 680 685 690 gaa tgc ata ctg cct gaa gga agaaca ggc gtc aca cag ata gga tat 2229 Glu Cys Ile Leu Pro Glu Gly Arg ThrGly Val Thr Gln Ile Gly Tyr 695 700 705 tgt gtg caa gac aca acc tct gccaat act acc ctt gtt cat cag acc 2277 Cys Val Gln Asp Thr Thr Ser Ala AsnThr Thr Leu Val His Gln Thr 710 715 720 aca cct tca cat gta atg cca ccaaat cac cac caa tta gca ttt aat 2325 Thr Pro Ser His Val Met Pro Pro AsnHis His Gln Leu Ala Phe Asn 725 730 735 740 tat caa gaa tta gaa cat ttacag act gtg aaa aac att tca cct tta 2373 Tyr Gln Glu Leu Glu His Leu GlnThr Val Lys Asn Ile Ser Pro Leu 745 750 755 caa att ctg cct ccc tca ggtgat tct gaa cag ctc tca aat ggc ata 2421 Gln Ile Leu Pro Pro Ser Gly AspSer Glu Gln Leu Ser Asn Gly Ile 760 765 770 act gtg atg cat cca tct ggtgat agt gac aca acg atg tta gaa tct 2469 Thr Val Met His Pro Ser Gly AspSer Asp Thr Thr Met Leu Glu Ser 775 780 785 gaa tgt caa gct cct gta cagaag gat ata aag att aag aat gca gat 2517 Glu Cys Gln Ala Pro Val Gln LysAsp Ile Lys Ile Lys Asn Ala Asp 790 795 800 tca tgg aaa agt tta ggc aaacca gtg aaa cca tca ggt gta atg aaa 2565 Ser Trp Lys Ser Leu Gly Lys ProVal Lys Pro Ser Gly Val Met Lys 805 810 815 820 tcc tca gat gag ctc ttcaac caa ttt aga aaa gca gcc ata gaa aag 2613 Ser Ser Asp Glu Leu Phe AsnGln Phe Arg Lys Ala Ala Ile Glu Lys 825 830 835 gaa gta aaa gct cgg acacag gaa ctc ata cgg aag cat ttg gaa caa 2661 Glu Val Lys Ala Arg Thr GlnGlu Leu Ile Arg Lys His Leu Glu Gln 840 845 850 aat aca aag gaa cta aaagca tct caa gaa aat cag agg gat ctt ggg 2709 Asn Thr Lys Glu Leu Lys AlaSer Gln Glu Asn Gln Arg Asp Leu Gly 855 860 865 aat gga ttg act gta gaatct ttt tca aat aaa ata caa aac aag tgc 2757 Asn Gly Leu Thr Val Glu SerPhe Ser Asn Lys Ile Gln Asn Lys Cys 870 875 880 tct gga gaa gag cag aaagaa cat ccg cag tca tca gaa gct caa gat 2805 Ser Gly Glu Glu Gln Lys GluHis Pro Gln Ser Ser Glu Ala Gln Asp 885 890 895 900 aaa tcc aaa ctc tggctt ctc aaa gac cgt gat tta gcc agg ccg aaa 2853 Lys Ser Lys Leu Trp LeuLeu Lys Asp Arg Asp Leu Ala Arg Pro Lys 905 910 915 gaa caa gag agg aggagg aga gaa gcc atg gtg ggt acc att gat atg 2901 Glu Gln Glu Arg Arg ArgArg Glu Ala Met Val Gly Thr Ile Asp Met 920 925 930 acc ctt caa agt gacatt atg aca atg ttt gaa aac aac ttt gat 2946 Thr Leu Gln Ser Asp Ile MetThr Met Phe Glu Asn Asn Phe Asp 935 940 945 taaaactcag tttttaaattaaccatccac ttaaaatgaa tggtaaaaga tcaaaatgca 3006 tatggtaaaa tgattgctttcagataacaa gataccaatc ttatattgta ttttgactgc 3066 tctaaaatga ttaaacagttttcacttaca aaaaaaaa 3104 3 22 DNA Artificial Sequence syntheticallygenerated primer 3 aatgtctctg ccaagtcgac aa 22 4 22 DNA ArtificialSequence synthetically generated primer 4 agcatccaca ggacgttgaa ag 22 515 PRT Homo sapiens 5 Cys Ser Glu Ile Leu Lys Glu Met Leu Ala Lys LysHis Phe Ser 1 5 10 15 6 11 PRT Homo sapiens 6 Tyr Tyr Thr Ile Ile LysAsn Pro Met Asp Leu 1 5 10 7 16 PRT Homo sapiens 7 Tyr Asn Lys Pro GlyAsp Asp Ile Val Leu Met Ala Gln Ala Leu Glu 1 5 10 15 8 15 PRT Homosapiens 8 Cys Ser Glu Ile Leu Lys Glu Met Leu Ala Lys Lys His Phe Ser 15 10 15 9 10 PRT Homo sapiens 9 Tyr Asp Val Val Lys Asn Arg Met Asp Leu1 5 10 10 7 PRT Homo sapiens 10 Tyr Lys Asp Ala Tyr Ser Phe 1 5 11 17PRT Homo sapiens 11 Tyr Asn Pro Pro Asp His Glu Val Val Thr Met Ala ArgMet Leu Gln 1 5 10 15 Asp 12 8 PRT Homo sapiens 12 Asn Tyr Asp Glu LysArg Gln Leu 1 5 13 15 PRT Homo sapiens 13 Tyr Leu His Lys Val Val MetLys Ala Leu Trp Lys His Gln Phe 1 5 10 15 14 11 PRT Homo sapiens 14 TyrHis Lys Ile Ile Lys Gln Pro Met Asp Met 1 5 10 15 16 PRT Homo sapiens 15Tyr Asn Lys Pro Thr Asp Asp Ile Val Leu Met Ala Gln Thr Leu Glu 1 5 1015 16 15 PRT Homo sapiens 16 Cys Asn Gly Ile Leu Lys Glu Leu Leu Ser LysLys His Ala Ala 1 5 10 15 17 10 PRT Homo sapiens 17 Tyr His Asp Ile LysHis Pro Met Asp Leu 1 5 10 18 7 PRT Homo sapiens 18 Tyr Arg Asp Ala GlnGlu Phe 1 5 19 17 PRT Homo sapiens 19 Tyr Asn Pro Pro Asp His Asp ValVal Ala Met Ala Arg Lys Leu Gln 1 5 10 15 Asp 20 8 PRT Homo sapiens 20Ser Tyr Asp Glu Lys Arg Gln Leu 1 5 21 15 PRT Drosophila melanogaster 21Tyr Ile Leu Lys Thr Val Met Lys Val Ile Trp Lys His His Phe 1 5 10 15 2211 PRT Drosophila melanogaster 22 Tyr His Lys Ile Ile Lys Gln Pro MetAsp Met 1 5 10 23 16 PRT Drosophila melanogaster 23 Tyr Asn Lys Pro GlyGlu Asp Val Val Val Met Ala Gln Thr Leu Glu 1 5 10 15 24 15 PRTDrosophila melanogaster 24 Cys Asn Glu Ile Leu Lys Glu Leu Phe Ser LysLys His Ser Gly 1 5 10 15 25 10 PRT Drosophila melanogaster 25 Tyr HisAsp Ile Lys His Pro Met Asp Leu 1 5 10 26 7 PRT Drosophila melanogaster26 Tyr Gln Ser Ala Pro Glu Phe 1 5 27 17 PRT Drosophila melanogaster 27Tyr Asn Pro Pro Asp His Asp Val Val Ala Met Gly Arg Lys Leu Gln 1 5 1015 Asp 28 8 PRT Drosophila melanogaster 28 Ser Tyr Asp Glu Lys Arg GlnLeu 1 5

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence having at least 60% identity to SEQ ID NO: 1,wherein the polypeptide regulates transcription of a gene and comprisesa bromodomain, but lacks an ATP-binding domain.
 2. The polypeptide ofclaim 1, wherein the amino acid sequence has at least 70% identity toSEQ ID NO:1.
 3. The polypeptide of claim 1, wherein the amino acidsequence has at least 80% identity to SEQ ID NO:
 1. 4. The polypeptideof claim 1, wherein the amino acid sequence has at least 90% identity toSEQ ID NO:
 1. 5. A substantially pure polypeptide comprising SEQ IDNO:1.
 6. A substantially pure polypeptide comprising the amino acidsequence of SEQ ID NO:1, with up to 30 conservative amino acidsubstitutions, wherein the polypeptide regulates transcription of a geneand comprises a bromodomain, but lacks an ATP-binding domain.
 7. Asubstantially pure polypeptide encoded by a nucleic acid that hybridizesunder highly stringent conditions to a probe consisting of SEQ ID NO:2,wherein the polypeptide regulates transcription of a gene and comprisesa bromodomain, buts lacks an ATP-binding domain.
 8. A substantially purepolypeptide consisting of SEQ ID NO:1.
 9. A substantially purepolypeptide comprising an amino acid sequence having at least 60%identity to SEQ ID NO:1, wherein the polypeptide regulates transcriptionof a gene, comprises a bromodomain, and does not bind ATP.
 10. Thepolypeptide of claim 9, wherein the amino acid sequence has at least 70%identity to SEQ ID NO:1.
 11. The polypeptide of claim 9, wherein theamino acid sequence has at least 80% identity to SEQ ID NO:1.
 12. Thepolypeptide of claim 9, wherein the amino acid sequence has at least 90%identity to SEQ ID NO:1.
 13. A substantially pure polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, with up to 30 conservative aminoacid substitutions, wherein the polypeptide regulates transcription of agene, comprises a bromodomain, and does not bind ATP.
 14. Asubstantially pure polypeptide encoded by a nucleic acid that hybridizesunder highly stringent conditions to a probe consisting of SEQ ID NO:2,wherein the polypeptide regulates transcription of a gene, comprises abromodomain, and does not bind ATP.
 15. A substantially pure polypeptidecomprising an amino acid sequence having at least 60% identity to SEQ IDNO:1, wherein the polypeptide regulates transcription of a gene andcomprises a bromodomain, but lacks a catalytic lysine.
 16. Thepolypeptide of claim 15, wherein the amino acid sequence has at least70% identity to SEQ ID NO:1.
 17. The polypeptide of claim 15, whereinthe amino acid sequence has at least 80% identity to SEQ ID NO:1. 18.The polypeptide of claim 15, wherein the amino acid sequence has atleast 90% identity to SEQ ID NO:1.
 19. A substantially pure polypeptidecomprising the amino acid sequence of SEQ ID NO:1, with up to 30conservative amino acid substitutions, wherein the polypeptide regulatestranscription of a gene and comprises a bromodomain, but lacks acatalytic lysine.
 20. A substantially pure polypeptide encoded by anucleic acid that hybridizes under highly stringent conditions to aprobe consisting of SEQ ID NO:2, wherein the polypeptide regulatestranscription of a gene and comprises a bromodomain, but lacks acatalytic lysine.
 21. A substantially pure polypeptide comprising anamino acid sequence having at least 60% identity to SEQ ID NO:1, whereinthe polypeptide regulates transcription of a gene and comprises abromodomain, but lacks kinase activity.
 22. The polypeptide of claim 21,wherein the amino acid sequence has at least 70% identity to SEQ IDNO:1.
 23. The polypeptide of claim 21, wherein the amino acid sequencehas at least 80% identity to SEQ ID NO:1.
 24. The polypeptide of claim21, wherein the amino acid sequence has at least 90% identity to SEQ IDNO:1.
 25. A substantially pure polypeptide comprising the amino acidsequence of SEQ ID NO:1, with up to 30 conservative amino acidsubstitutions, wherein the polypeptide regulates transcription of a geneand comprises a bromodomain, but lacks kinase activity.
 26. Asubstantially pure polypeptide encoded by a nucleic acid that hybridizesunder highly stringent conditions to a probe consisting of SEQ ID NO:2,wherein the polypeptide regulates transcription of a gene and comprisesa bromodomain, but lacks kinase activity.
 27. A method of screening fora compound that binds to a polypeptide, the method comprising providinga polypeptide comprising an amino acid sequence, having at least 60%identity to SEQ ID NO:1, wherein the polypeptide regulates genetranscription and comprises a bromodomain, but lacks an ATP-bindingdomain; contacting a test compound with the polypeptide; determiningwhether the test compound has bound to the polypeptide, and selecting acompound that binds to the polypeptide.
 28. A method of screening for acompound that binds to a polypeptide, the method comprising: providingthe polypeptide of claim 2; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 29.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 3; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 30. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 4; contacting a test compound with the polypeptide; determiningwhether the test compound has bound to the polypeptide, and selecting acompound that binds to the polypeptide.
 31. A method of screening for acompound that binds to a polypeptide, the method comprising: providingthe polypeptide of claim 5; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 32.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 6; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 33. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 7; contacting a test compound with the polypeptide; determiningwhether the test compound has bound to the polypeptide, and selecting acompound that binds to the polypeptide.
 34. A method of screening for acompound that binds to a polypeptide, the method comprising: providingthe polypeptide of claim 9; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 35.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 10; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 36. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 11; contacting a test compound with the polypeptide;determining whether the test compound has bound to the polypeptide, andselecting a compound that binds to the polypeptide.
 37. A method ofscreening for a compound that binds to a polypeptide, the methodcomprising: providing the polypeptide of claim 12; contacting a testcompound with the polypeptide; determining whether the test compound hasbound to the polypeptide, and selecting a compound that binds to thepolypeptide.
 38. A method of screening for a compound that binds to apolypeptide, the method comprising: providing the polypeptide of claim13; contacting a test compound with the polypeptide; determining whetherthe test compound has bound to the polypeptide, and selecting a compoundthat binds to the polypeptide.
 39. A method of screening for a compoundthat binds to a polypeptide, the method comprising: providing thepolypeptide of claim 14; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 40.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 15; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 41. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 16; contacting a test compound with the polypeptide;determining whether the test compound has bound to the polypeptide, andselecting a compound that binds to the polypeptide.
 42. A method ofscreening for a compound that binds to a polypeptide, the methodcomprising: providing the polypeptide of claim 17, contacting a testcompound with the polypeptide; determining whether the test compound hasbound to the polypeptide, and selecting a compound that binds to thepolypeptide.
 43. A method of screening for a compound that binds to apolypeptide, the method comprising: providing the polypeptide of claim18; contacting a test compound with the polypeptide; determining whetherthe test compound has bound to the polypeptide, and selecting a compoundthat binds to the polypeptide.
 44. A method of screening for a compoundthat binds to a polypeptide, the method comprising: providing thepolypeptide of claim 19; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 45.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 20; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 46. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 21; contacting a test compound with the polypeptide;determining whether the test compound has bound to the polypeptide, andselecting a compound that binds to the polypeptide.
 47. A method ofscreening for a compound that binds to a polypeptide, the methodcomprising: providing the polypeptide of claim 22; contacting a testcompound with the polypeptide; determining whether the test compound hasbound to the polypeptide, and selecting a compound that binds to thepolypeptide.
 48. A method of screening for a compound that binds to apolypeptide, the method comprising: providing the polypeptide of claim23; contacting a test compound with the polypeptide; determining whetherthe test compound has bound to the polypeptide, and selecting a compoundthat binds to the polypeptide.
 49. A method of screening for a compoundthat binds to a polypeptide, the method comprising: providing thepolypeptide of claim 24; contacting a test compound with thepolypeptide; determining whether the test compound has bound to thepolypeptide, and selecting a compound that binds to the polypeptide. 50.A method of screening for a compound that binds to a polypeptide, themethod comprising: providing the polypeptide of claim 25; contacting atest compound with the polypeptide; determining whether the testcompound has bound to the polypeptide, and selecting a compound thatbinds to the polypeptide.
 51. A method of screening for a compound thatbinds to a polypeptide, the method comprising: providing the polypeptideof claim 26; contacting a test compound with the polypeptide;determining whether the test compound has bound to the polypeptide, andselecting a compound that binds to the polypeptide.